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TISSUE RNA PURIFICATION KIT
Product Manual
26-004 ...............5 preps
26-010 ...............50 preps
26-010B ............50 preps
P/N 03-293-4 REV. E
Omni Tissue RNA Purification Kit
TABLE OF CONTENTS
1. Introduction .............................................................................. Pg1
2. Overview ..................................................................................... Pg1
3. Kit Components ..................................................................... Pg2
4. Storage and Stability ........................................................... Pg2
5. Illustrated Protocols ............................................................. Pg3
6. Before Starting ......................................................................... Pg4
7. Preparing Reagents .............................................................. Pg5
8. Omni Tissue RNA Kit Instructions ................................ Pg5
9. Integrity of RNA ...................................................................... Pg7
10. Quantification and Storage of RNA ......................... Pg7
11. Troubleshooting Guide ................................................... Pg8
Omni Tissue RNA Purification Kit
Omni Tissue RNA Purification Kit Omni Tissue RNA Purification Kit
INTRODUCTION
The Omni Tissue RNA Kit is designed for isolation of total RNA from animal tissues. The Omni Tissue RNA Kit allows simultaneous processing of multiple tissue samples in less than 60 minutes. This procedure completely removes contaminants and enzyme inhibitors making RNA isolation fast, convenient, and reliable.
OVERVIEW
The Omni Tissue RNA Kit uses the reversible binding properties of the Omni
RNA Mini Column matrix, a silica-based material. The sample is lysed first under highly denaturing buffer conditions so that RNase’s are inactivated and the intact Tissue RNA is protected from degradation. Cell debris is pelleted by centrifugation. After adding ethanol to the cleared lysate, the sample is loaded into the Omni RNA Mini column. With a brief centrifugation step, the samples pass through the column and the RNA binds to the Omni RNA Mini Column matrix. Trace DNA that may co-purify with RNA can be removed by an optional
DNase treatment step on the RNA spin column. After two wash steps, purified total RNA is eluted with RNase-free water.
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Omni Tissue RNA Purification Kit
KIT COMPONENTS
Product Number
Purifications
Omni RNA Mini Columns
2 mL Collection Tubes
RLB Buffer
26-004 26-010 26-010B
5 50 50
5
15
5 mL
50
150
40 mL
50
150
40 mL
RW1 Buffer
RW2 Buffer
5 mL
5 mL
50 mL 50 mL
12 mL 12 mL
DEPC Water 1.5 mL 20 mL 20 mL
2 mL bead kit 2.8 mm ceramic 5 50
Antifoam
Instruction Manual
1 mL 1 mL
STORAGE AND STABILITY
All components in the Omni Tissue RNA Kit can be stored at room temperature for 12 months. During shipping and storage, precipitate may form in the RLB Buffer. Simply warm to 37 o C to dissolve.
Omni Tissue RNA Purification Kit
Omni Tissue RNA Purification Kit Omni Tissue RNA Purification Kit
ILLUSTRATED PROTOCOLS
Lyse and homogenize tissues
Adjust the binding conditions
Transfer lysate into a
RNA Mini Column to bind RNA
Wash column x 3
Dry the column
Elute RNA
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Omni Tissue RNA Purification Kit
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BEFORE STARTING
Please take a few minutes to read this booklet thoroughly and become familiar with the protocol. Prepare all of the materials required before starting to minimize RNA degradation.
Materials supplied by user
• 96-100% ethanol
• RNase-Free DNase I (optional)
• 2-Mercaptoethanol (βME)
• RNase-free filter pipette tips
• Bead Mill Homogenizer or Vortexer
• Centrifuge capable of 10,000 x g
• Water bath or heat block preset at 55 o C
• Disposable gloves
• 70% ethanol in DEPC treated water
Although the binding capacity of the Omni RNA mini column is around 100
µg, the maximum amount of starting material depends on the type of tissue being processed and the corresponding RNA content.
It is essential to begin with the correct amount of tissue to get optimal RNA yield and purity with the Omni RNA Mini Column. For the first time user, we recommend to use less than 10 mg of tissue. Depending on the yield and purity obtained, it may be possible to increase the starting material to 30 mg.
• Whenever working with RNA, always wear latex gloves to minimize RNase
contamination. Use only clean RNase-free disposable plastic pipette tips
when using the supplied reagents.
• During the procedure work carefully but quickly.
• Under cool ambient conditions, crystals may form in RLB Buffer. This is
normal and the bottle should be warmed to redissolve the salt.
• 2-mercaptoethanol (βME) is key in denaturing RNase’s
and must be added to an aliquot of RLB Buffer before use. Add 20 µl of
2-mercaptoethanol per 1 mL of RLB Buffer. This mixture can be stored for
1 week at room temperature.
• All centrifugation steps must be carried out at 22 o C-25 o C
Note: Equilibrate samples and RLB buffer to room temperature before start. All steps must be carried out at room temperature. Work quickly, but carefully.
Omni Tissue RNA Purification Kit
Omni Tissue RNA Purification Kit Omni Tissue RNA Purification Kit
PREPARING REAGENTS
Dilute RW2 Buffer with 100% ethanol as follows and store at room temperature.
Kit 100% ethanol to be added
26-004
26-010 and 26-010B
20 mL
48 mL
OMNI TISSUE RNA PURIFICATION KIT DIRECTIONS
26-010 - Rotor Stator or Manual
Tissue Disruption
26-010B - Bead Mill
Homogenization
1. Excise the tissue sample from the
animal or from storage.
2. Weigh 10-30 mg of tissue
A. Mortar and Pestle processing -
Freeze sample in liquid
nitrogen. Grind sample to
fine powder under liquid
nitrogen. Transfer powder
to a clean nuclease free 1.5 mL
microcentrifuge tube (not
provided). Add appropriate
amount of RLB Buffer/βME
from table below.
B. Rotor-Stator Homogenization -
Transfer sample to nuclease
free 2-5 mL tube (not
provided). Add appropriate
amount of RLB Buffer/βME
from table below. Process
sample until a full homogenate
is achieved. Most tissues can
be fully homogenized in less
than 1 minute. Plastic Omni-Tip
disposable generator probes
(Cat# 30750H) are recommended
for this process.
1. Excise the tissue sample from the
animal or from storage
2. Weigh 10-30 mg of tissue and
place into a 2 mL tube
containing 2.8 mm ceramic beads.
3. Add appropriate amount of RLB
Buffer/βME from table below
and 10 uL antifoam reagent.
4. Dissociate tissues on a bead mill.
If a bead mill is not available
the tissue can be processed on a
vortexer at maximum speed for
10 minutes.
Amount of Tissue Amount of RLB Buffer
< 15 mg 350 µL
20 -30 mg 700 µL
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Omni Tissue RNA Purification Kit
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Note: Bead Mill speed/power and time settings should be adjusted based on the equipment manufacturer’s recommendations for the specific sample type. When using mechanical methods of homogenization care must be taken to not over process as this could lead to RNA shearing.
5. Centrifuge at 10,000 x g for 5 minutes to pellet debris.
6. Transfer the cleared supernatant to a nuclease-free 1.5 mL
microcentrifuge tube (Not provided)
Note: In some preparations, a fatty upper layer will form after centrifugation. Transfer of any of this layer may reduce RNA yield or clog the column.
7. Add 1 volume of 70% ethanol. Vortex for 10 seconds to mix thoroughly.
Do not centrifuge.
Note: A precipitate may form at this point. This will not interfere with RNA purification. If any sample has lost its volume during homogenization, adjust the volume of ethanol accordingly.
8. Insert an Omni RNA Mini Column into a 2 mL Collection Tube.
9. Carefully transfer 700 µL of sample from step 7 (including any precipitate)
to the Omni RNA Mini Column.
10. Spin at 10,000 x g for 1 minute at room temperature. Discard the flow-
through and re-use the collection tube.
11. Repeat steps 9 and 10 until the entire sample has been transferred.
12. Pipet 500 µL of RW1 Buffer into the column.
13. Centrifuge at 10,000 x g for 30 seconds. Discard the flow-through and
collection tube.
14. Place the column into a new collection tube (provided). Pipet 500 µL of
RW2 Buffer into the column.
Note: RW2 Buffer must be diluted with absolute ethanol before use.
Refer to the label on the bottle for instruction.
15. Centrifuge at 10,000 x g for 1 minute.
16. Discard the flow-through and re-use the collection tube.
17. Add another 500 µL of RW2 Buffer to the column
18. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and
reuse collection tube.
Omni Tissue RNA Purification Kit
Omni Tissue RNA Purification Kit Omni Tissue RNA Purification Kit
19. Centrifuge at 10,000 x g for 2 minutes to completely dry the column.
20. Elution of RNA: Place the Omni RNA Mini Column onto a 1.5 mL
RNase-free microcentrifuge tube and add 50 µL of DEPC-treated water
directly onto the center of Omni RNA Mini Column. Centrifuge at
10,000 x g for 2 minutes to elute the RNA.
21. If the expected RNA yield is greater than 30 µg, repeat the elution step
as described with a second volume of DEPC-treated water, collect the
second elution with the same collection tube. To obtain a higher
concentration of RNA, the second elution can be performed using the
first elute (from step 16). Heating the DEPC water to 70 o C before adding
to the column and incubating the column after the addition of DEPC
water for 5 minutes at room temperature can increase RNA yield as well.
INTEGRITY OF RNA
Run a denatured gel to determine the integrity and the size distribution of the RNA. The respective ribosomal RNA bands should appear as sharp and clear bands on the gel. If the ribosomal RNA bands in a given lane are not sharp and it shows smearing towards smaller sized RNA, it is very likely that the isolated RNA suffered major degradation during isolation procedure
QUANTIFICATION AND STORAGE OF RNA
To determine the concentration and purity of RNA, measure the absorbency at 260nm and 280nm in a spectrophotometer. One OD unit measured at
260nm corresponds to 40 µg of RNA per ml. DEPC water is slightly acidic and can dramatically lower absorbance values. We suggest that you dilute the sample in a buffered solution (TE) for spectrophotometric analysis.
The A
260
/A
280
ratio of pure nucleic acids is 2.0, while for pure protein it is approximately 0.6. A ratio of 1.8-2.1 corresponds to 90%-100% pure nucleic acid. Store RNA samples at -80 o C. Under such conditions RNA is stable for more than a year.
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Omni Tissue RNA Purification Kit
TROUBLE SHOOTING GUIDE
Problem Cause
Little or no RNA eluted
Clogged Column
Degraded RNA
Problems in downstream applications
DNA Contamination
Low Abs ratio
RNA remains on the column
Column is overloaded
Incomplete homogenization
Source
RNase Contamination
Salt carry-over during elution
Co-purification of DNA
RNA diluted in acidic buffer or water.
Suggestion
• Repeat elutions
• Preheat DEPC water to 70°
prior to elution
• Incubate column for 5 min
with water prior to
centrifugation.
• Reduce quantity of starting
material
• Completely homogenize sample
• Increase configuration time
• Reduce amount of starting
material.
• Do not freeze and thaw sample
more than once
• Follow protocol closely and
work quickly.
• Ensure not to introduce RNase
during the procedure
• Check buffers for RNase
contamination
• Ensure RW2 buffer concentrate
has been diluted with 4
volumes of 100% ethanol as
indicated on botltle.
• RW2 buffer must be stored
and used at room temperature.
• Repeat with RW2 buffer.
• Digest with RNase free Dnase
after elution and inactive at
65°C for 5min in the presence
of EDTA.
• DEPC water is acidic
Omni Tissue RNA Purification Kit
Omni Tissue RNA Purification Kit
NOTES
Omni Tissue RNA Purification Kit
935-C Cobb Place Blvd
Kennesaw, GA 30144
Phone: 800.776.4431
Fax: 770. 421.0206
www.omni-inc.com

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